Hedgehog (Hh) signaling plays a prominent role in embryogenesis, and its deregulation is implicated in tumorigenesis. Cellular responses to the Hedgehog (Hh) signal are controlled by two transmembrane proteins: the tumor suppressor Patched (PTCH) and the proto-oncogene Smoothened (SMO). In the absence of secreted Hh proteins, PTCH actively silences intracellular signaling by inactivating SMO. During physiologic signaling, Hh proteins bind and inactivate PTCH, which alleviates PTCH-mediated suppression of SMO. SMO activation triggers a series of intracellular events, culminating in expression of Hh target genes through the action of the Gli family of transcription factors. Gli1, Gli2 and Gli3, and their Drosophila homolog, Cubitus interruptus (Ci), are zinc finger transcription factors that are downstream effectors of Hh signaling. In the absence of Hh signaling, Ci is truncated at the carboxyl terminal domain to form a truncated repressor protein, whereas Hh activation leads to accumulation of transcriptionally active, full length Ci. The situation with mammalian Gli proteins is more complex. Gli3 functions primarily as a C-terminally truncated repressor, but full-length Gli3 protein accumulates in cells responding to Hh. Gli1, on the other hand, appears to modulate gene expression by acting primarily as a transcriptional activator, but Gli1 mutant mice are phenotypically normal, arguing against an essential function for this protein during development or postnatal life. Gli2 appears to be the major nuclear effector of Hh signaling in vivo and functions primarily as a transcriptional activator. However, little is known about the molecular mechanisms regulating Gli2 expression at the protein level.
The Hh signaling pathway is deregulated in many human malignancies, including skin cancer, such as, basal cell carcinoma (BCC), medulloblastoma, glioblastoma, rhabdomyosarcoma, lung, prostate, breast, and some gastrointestinal cancers. Recent studies have stressed the importance of Hh signaling in human prostate cancer. Elevated Hh signaling pathway activity may distinguish metastatic from localized prostate cancer, and pathway manipulation can modulate invasiveness and metastasis. In contrast to BCCs and mebulloblastomas, which are associated with inactivating mutations in PTCH or gain-of-function mutations in SMO, aberrant Hh signaling in prostate cancers appears to be the result of constitutive overexpression of Sonic hedgehog (SHH). Hence, the growth of many of the prostate cancer cells is inhibited by Hh-neutralizing antibody. Furthermore, cyclopamine, a steroidal alkaloid that interacts with SMOH directly thus inhibiting Hh signaling, was shown to induce apoptosis and inhibit proliferation of prostate cancer cells in vivo as well as in vitro.
The ubiquitin-proteasome pathway is essential for degradation of proteins regulating growth and cell cycle progression. Ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin-ligase (E3) sequentially tag proteins for ubiquitination and proteasomal degradation. SCF E3 ubiquitin ligases are composed of Skp1, Cul1, Roc1 and F box proteins, where F box proteins are substrate recognizing subunits. Beta-transducin repeat-containing F box proteins (β-TrCP) recognize substrates phosphorylated within the DSG(X)2+nS destruction motifs. SCFβ-TrCP E3 ligases ubiquitinate specifically phosphorylated substrates and play a pivotal role in the regulation of cell division and various signal transduction pathways, which, in turn, are essential for many aspects of tumorigenesis. Genetic data have suggested that Drosophila slimb protein (orthologue of mammalian β-TrCP) is involved in proteolytic processing of Ci155 to Ci75. However, there is no biochemical evidence that Slimb/β-TrCP proteins are involved in ubiquitination and degradation of Ci/Gli transcription factors.
Stabilization of the transcription factor Gli2 has been suggested as a key event in the transduction of Hh signals. The potential role of Gli2 in the development of BCC has been well documented. Gli2 is over-expressed in the majority of human BCCs, and skin-targeted over-expression of Gli2 in transgenic mice leads to the development of multiple BCCs. There is growing evidence that the transcriptional regulation of some Hh target genes, including Gli1, E2F1, Bcl2, etc., are Gli2 dependent. The promoter of one such gene, Bcl2, is regulated preferentially by Gli2.
As disclosed herein, the inventors have found that SCFβ-TrCP E3 ubiquitin ligase is responsible for Gli2 degradation. β-TrCP2 binds wild type Gli2 and promotes its ubiquitination, which can be altered by a single amino acid substitution in the DSGX binding site of Gli2 with β-TrCP2. As disclosed here, mutating residues within the DSGX binding motif, inhibits binding of β-TrCP2 to Gli2, increasing Gli2 half-life and promoting Gli2 dependent transcription. The inventors further show that Gli2 is over-expressed in prostate cancer cell lines and primary tumors. Thus, expression of Gli2 with altered binding to β-TrCP2 provides a model for investigating diseases identified with Gli2 overexpression and for identifying agents that interfere with Gli2 dependent transcription and/or diseases resulting therefrom.